Int.J.Curr.Microbiol.App.Sci (2014) 3(7) 812-816
ISSN: 2319-7706 Volume 3 Number 7 (2014) pp. 812-816
http://www.ijcmas.com
Original Research Article
Mutagenicity of fungicide Mancozeb by Salmonella Reverse Mutation assay
Nimisha D. Patel1*, Nilofar M. Shaikh1, Shyama A. Mehta1 and Rajendra M. Nagane2
1
C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Maliba campus,
Bardoli, Surat 394350 Gujarat, India
2
Jay Research Foundation, N.H. No. 8 Valvada - 396 108, Dist. Valsad, Gujarat, India
*Corresponding author
ABSTRACT
Keywords
Mancozeb,
Auxotrophic,
Salmonella
typhimurium,
Mutagenicity,
Ames test
Mancozeb, a fungicide of class dithiocarbomates, was studied for its mutagenic
potential. Ames Salmonella mutagenicity assay was used in which mancozeb at
different concentrations was checked for its ability to induce reverse mutation at
histidine locus in three strains of Salmonella typhimurium (TA98,TA100 and
TA102) in the presence and in absence of metabolic activating system (S9)
containing mammalian microsomal enzymes. Based on Cytotoxicity test 0.002 and
0.001 mg of mancozeb/plate was selected as the highest concentration to be tested
in the mutagenicity test in the absence and in the presence of metabolic activation,
respectively. In case of mean, number of revertant colonies at 0.002 mg plate-1 in
absence of metabolic activation for TA98 was 12, TA100 was 92 and TA102 was
226 which were below positive control and number of revertants at 0.001mg plate-1
in presence of metabolic activation for TA98 was 12, TA100 was 105 and TA102
was 282 which were also less as compared to positive control. Thus, mancozeb up
to concentration of 0.002 mg plate-1 in absence of metabolic activation and 0.001
mg plate-1 in presence of metabolic activation (5% v/v S9 mix), was non
mutagenic to all three strains of Salmonella typhimurium when tested under
specific conditions. There was no significant increase in number of revertant
colonies accomplished that mancozeb with this concentration can be considered as
non mutagenic.
Introduction
and were proved to be effective and also
ecofriendly (Gondal et al., 2012). It acts a as
fungicide by inhibiting enzyme activity in
fungi by forming a complex with metalcontaining enzymes including those
involved in production of adenosine
triphosphate (ATP). However, it is listed as
a cancer-causing chemical by California's
Office of Environmental Health Hazard
Mancozeb is marketed by the trade names
Dithane, Maneb, Nemispot, and Manzane.
The fungicide mancozeb is one group of
pesticides
known
as
ethylene
bisdithiocarbomate (EBDCs) registered for
use on a variety of vegetable, fruit, nut and
grain crops. (Hayes et al., 1990). Different
doses of mancozeb were checked for its
effect on Alternaria leaf blight of potatoes
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Int.J.Curr.Microbiol.App.Sci (2014) 3(7) 812-816
Assessment (OEHHA) under proposition 65
dated July 26, 2013. (http:// oehha. ca.gov/
prop65/prop65_list/Newlist.html) Thus, it
can be considered as multipotent
carcinogenic agent (Belpoggi etal., 2002). It
is reported to alter structural and functional
changes in thyroid of rats and also affect
level of glycogen, proteins and lipids in
testis, liver and kidney (Kackar et al., 1997;
Ksheerasagar et al., 2010; and Axelstad et
al., 2011). Mancozeb induces genotoxicity
and
apoptosis in cultured human
lymphocytes
(CHLs).
The
Studies
conducted on micronuclei and chromosome
abbration in blood of healthy donors were
found to induce apoptosis in CHLs
(Srivastava et al., 2012).
strains of Salmonella typhimurium (TA98,
TA100 and TA1537), in the presence and
absence of an exogenous metabolic
activation
system
(S9)
containing
mammalian microsomal enzymes
Materials and Methods
Salmonella typhimurium strains (TA98,
TA100 and TA102) were procured from
Toxicology Department, Jai Research
Foundation. The strains were checked for
histidine dependency, biotin dependency,
histidine/biotin dependency, Rfa mutation,
ampicillin and tetracycline resistance. Rat
liver S9 fractions were prepared from
Phenobarbital-treated
rats.
Plate
incorporation assay was done according to
the standard protocols (Mortelmans et al.,
2000) Cytotoxicity test was performed to
select
treatment
concentration
for
mutagenicity test. Mancozeb was dissolved
in DMSO (dimethyl sulfoxide) and diluted
prior to treatment according to test
concentration. A liver S9 mixture was added
at the concentration of 0.5 ml per plate.
Ames Salmonella Test is used for assaying
mutagenicity. It is simple, inexpensive and
extremely sensitive test which makes use of
a rat or human liver homogenate for
carcinogen activation (thus supplying
mammalian metabolism) and a set of
Salmonella histidine mutants for mutagen
detection. It is an initial screening method
for detecting carcinogenicity of chemicals
(Mortelmans et al., 2000). Muhammed et al.
(2003) reported that mancozeb had some
effect on the hematological parameters of
rainbow trout. The RBC/WBC level may be
increased due to decrease in WBC count
during the study.
Toxicity of mancozeb was determined by
plating 100 l of diluted test substance with
100 l overnight grown standard bacterial
cultures and top agar enriched with 0.5mM
histidine /biotin. A positive control 2Nitrofluorene (7.5 g plate-1) for TA98,
Sodium azide (5 g/plate) for TA100,
Mitomycin-C (0.5 g plate-1) for TA102
and positive control devoid of 2aminoanthracene for all the three strains in
absence of metabolic activation was used.
Along with it positive Control 2Aminoanthracene (10 g plate-1) for TA102
and (5 g plate-1) for TA98 and TA100 in
presence of metabolic activation was used to
check mutagenicity of the strains. A dose
response increase in number of revertants
over the positive control value was taken as
the affirmation for mutagenicity.
Investigations regarding its degradation in
tomatoes homogenate were studied to
prevent the risk of it and its metabolites
exposure to diet (Certel et al., 2011). It also
interferes with carbon and nitrogen
assimilation and thus in carbon and nitrogen
cycle and may further lead to toxicity in
inbuilt soil microorganisms (Cernohlavkova
et al., 2008). Therefore, the aim of the
present study was to figure out non toxic
concentration of mancozeb and to evaluate
mancozeb for its ability to induce reverse
mutations at the histidine locus in three
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Int.J.Curr.Microbiol.App.Sci (2014) 3(7) 812-816
provide more sensitive assessment for
mutagenic activity. Also significant
inhibition in chromosome aberration was
found with mancozeb at 250 mg kg-1 b.wt.
in Swiss albino mice and no significant
effect in growth and reproduction in E.fetida
was found with mancozeb at recommended
dose (8mg kg-1) (Tripathi et al., 2011;
Vermeule et al., 2001), which may suggest
that concentration taken in Experiment
shows non mutagenicity. A similar class of
fungicide, maneb did not show double
increase in revertants in micronucleus and
chromosome aberration test and was proved
to be non-mutagenic. While another
fungicide, thiram in Ames test, proved lethal
at higher concentration to micro-organisms
(Andres et al., 1999).
Results and Discussion
Cytotoxic testpartial background bacterial
lawn inhibition, was observed at the
concentration of 0.00098 and 0.03907
mg/plate in the absence and presence of
metabolic activation (5% v/v S9 mix),
respectively. Reduction in the number of
revertant colonies was observed at the
concentration
of
0.0009765625
and
0.001953125 mg plate-1 in the absence and
presence of metabolic activation (5% v/v S9
mix), respectively. Hence, 0.002 and 0.001
mg of mancozeb/plate was selected as the
highest concentration.
In case of mean number of revertant
colonies obtained in the absence of
metabolic activation at 0.002 mg plate-1 for
TA98 was 12, TA100 was 92 and TA102
was 226 and in case of presence of
metabolic activation at 0.001mg plate-1 the
number of revertants obtained with TA98
was 12, TA100 was 105 and TA102 was
282 which are below positive control. So the
mean number of revertant colonies obtained
were far less as compared to positive
control. Thus, none of the plate
incorporation assays and linear regression
analysis tester strains used showed
significant increase in revertant numbers
(Table 1 and 2).
So it increase in number of revertants with
increase in dose concentration. However,
with evaluation of toxic effect of mancozeb
at concentration of 500 mg kg-1 body
weight. in rats showed toxicity in testis in
males and in female mice show gonaldal
toxicity which in case of female may be due
to hormonal reaction or its own toxicity
(Joshi et al., 2005; Baligar et al., 2001). This
correlation suggests that it may lead toxicity
with high dosage level . and also toxicity
varies with respect to specificity in rats,
human and other animals.
It is evident from the present study that
mancozeb exhibited weak mutagenic
response at tested dosage and if the similar
dose seems to be having fungicidal activity
we can use mancozeb at this concentration
as a fungicide. Further studies with different
metabolic activation system may give a
better understanding of the genotoxic
potential of mancozeb, as it is still used in
India.
It was observed that at 0.002 and 0.001 mg
of mancozeb/plate in the presence and
absence of metabolic activation, none of the
tester strain showed toxic effect and no
significant increase in the number of
revertants was observed when compared
with positive control. Thus, the true
mutagenic potential of mancozeb may be
masked by its toxic effect on the tester strain
used. Further modification of Ames test may
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Int.J.Curr.Microbiol.App.Sci (2014) 3(7) 812-816
Table.1 Mean count of His+ revertant colonies in negative control (NC) and positive control
(PC) and treatment plates in the absence of metabolic activation
Mancozeb
Concentration
(mg plate-1)
HIS+ revertant colonies/plate(Absence of metabolic activation)
TA98
TA100
TA102
Mean
SD
Mean
SD
Mean
SD
NC(DW)
26.00 ±1.41
132.00
±12.73
222.50
±3.54
0.0000625
21.50 ±4.95
81.50
±12.02
232.00
±7.07
0.000125
22.50 ±3.54
77.50
±0.71
243.00
±11.31
0.00025
18.50 ±4.95
102.50
±13.44
236.00
±1.41
0.0005
23.50 ±0.71
100.50
±14.85
231.00
±14.14
0.001
0.002
PC
PC-2Aa
20.00 ±0.00
12.00 ±4.24
495.00 ±42.45
-
115.00 ±26.87
92.00
±8.49
1263.50 ±232.64
143.5
±14.85
267.50
226.00
372.00
±33.23
±11.31
±46.67
SD = Standard Deviation, NC = Negative Control, PC = Positive Control, DW = Distilled
Water, - = Not applicable
Table.2 Mean count of His+ revertant colonies in negative, vehicle and positive controls and
treatment plates in the presence of metabolic activation (Trial I)
Mancozeb
Concentration
(mg plate-1)
HIS+ revertant colonies/plate(Presence of metabolic activation)
TA98
Mean
SD
TA100
Mean
SD
TA102
Mean
SD
0.00003125
21.00
±7.07
107.00
±7.07
271.50
±20.51
0.0000625
23.50
±0.71
94.50
±26.16
222.00
±14.14
0.000125
20.50
±4.95
85.50
±2.12
250.50
±33.23
0.00025
21.50
±0.71
106.50
±14.85
280.00
±5.66
0.0005
13.50
±0.71
89.00
±9.90
256.50
±3.54
0.001
PC
12.00 ±5.66
795.00 ±43.84
282.00
1526.50
±15.56
±71.42
105.50 ±3.54
1225.50 ±14.85
SD = Standard Deviation, NC = Negative Control, PC = Positive Control, DW = Distilled
Water
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Int.J.Curr.Microbiol.App.Sci (2014) 3(7) 812-816
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Acknowledgment
Authors are thankful to Dr. Pradeep B.
Deshmukh
(Head,
Department
of
Toxicology) for his assistance and for
providing research facility to carry out
present work.
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